The present invention concerns the analysis of mixtures of compounds. More particularly, the present invention involves tagging individual compounds with unique fluorescent markers having different fluorescence lifetimes. The analysis of the mixture is then accomplished by distinguishing individual compounds by their unique fluorescence lifetime.
In numerous fields, including organic chemistry, forensics, medical diagnosis and molecular biology there is a growing need for safe, efficient and cost-effective methods for identifying compounds of interest within a mixture of compounds. Mixtures of compounds frequently arise as the product of an organic synthetic cycle, during the isolation of a product of biological origin and during the chemical or enzymatic sequencing of polymeric compounds such as polypeptides, proteins, polysaccharides and nucleic acids.
Accurately determining nucleic acid base sequence is a prerequisite to further understanding the structure and function of the proteins produced by the encoded information. One such method, DNA sequencing, involves determining the order in which the nucleic acid bases are arranged within a length of DNA. Two DNA sequencing techniques which are widely known and in current use, are the chemical degradation procedure according to Maxam and Gilbert (Proc. Natl. Acad. Sci. USA 74:560 (1977)) and the enzymatic dideoxy chain termination method of Sanger et al (Proc. Natl. Acad. Sci. USA 74:5463 (1977)). Additionally, reference is made to, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (Supplement 37, current through 1997) (Ausubel), particularly, Chapter 7, which is incorporated herein by reference, for a description of DNA sequencing in general and various DNA sequencing techniques.
Traditional methods of DNA sequencing utilize a radiolabeled oligonucleotide primer to synthesize a nucleic acid having a sequence complementary to the sequence under analysis. Alternatively, a radiolabeled nucleotide is incorporated directly into the growing nucleic acid strand. Following synthesis, the radioactive nucleic acids are separated by a method such as gel electrophoresis and the positions of the nucleic acids are visualized by autoradiography. Although this technique provides sensitive detection, the use of radioisotopes and autoradiography requires extended exposure times and presents waste disposal problems.
Fluorescent-labeled oligonucleotide primers have been used in place of radiolabeled primers for sensitive detection of DNA fragments (U.S. Pat. No. 4,855,225 to Smith et al.). Additionally, DNA sequencing products can be labeled with fluorescent dideoxynucleotides (U.S. Pat. No. 5,047,519 to Prober et al.) or by the direct incorporation of a fluorescent labeled deoxynucleotide (Voss et al. Nucl. Acids Res. 17:2517 (1989)). As currently practiced, fluorescent sequencing reactions circumvent many of the problems associated with the use of radionuclides.
In an attempt to increase laboratory throughput and to further decrease exposure of laboratory workers to harmful reagents, various strategies have been developed. For example, robotic introduction of fluids onto microtiter plates is commonly performed to speed mixing of reagents and to enhance experimental throughput. More recently, microscale devices for high throughput mixing and assaying of small fluid volumes have been developed. For example, U.S. Ser. No. 08/761,575 entitled High Throughput Screening Assay Systems in Microscale Fluidic Devices by Parce et al. provides pioneering technology related to microscale fluidic devices, especially including electrokinetic devices. The devices are generally suitable for assays utilizing fluorophores which relate to the interaction of biological and chemical species, including enzymes and substrates, ligands and ligand binders, receptors and ligands, antibodies and antibody ligands, as well as many other assays. Because the devices provide the ability to mix fluidic reagents and assay mixing results in a single continuous process, and because minute amounts of reagents can be assayed, these microscale devices represent a fundamental advance for laboratory science.
The application of fluorogenic and non-fluorogenic assays utilizing fluorescent labels in flowing microfluidic systems are provided in Kopf-Sill et al. U.S. Ser. No. 09/093,542 xe2x80x9cApparatus and Methods For Correcting for Variable Velocity in Microfluidic Systems,xe2x80x9d filed Jun. 8, 1998. A fluorogenic assay is an assay in which a product of the assay emits a label distinct from those of the reactants of the assay. A non-fluorogenic assay is an assay in which the mobility of a product differs from those of labeled reactants (e.g., in a flowing electrokinetic system), but the emitted label is still the same as the label found on a reactant. Detection of non-fluorogenic assay products is possible in an electroosmotically driven microfluidic device using periodic injections of reaction mixture into a separation channel, in which reactants and products are separated by electrophoresis due to changes in the electrophoretic mobility resulting from the reaction (see also, A. R. Kopf-Sill, T. Nikiforov, L. Bousse, R. Nagel, and J. W. Parce, xe2x80x9cComplexity and performance of on-chip biochemical assays,xe2x80x9d in Proceedings of Micro- and Nanofabricated Electro-Optical Mechanical Systems for Biomedical and Environmental Applications, SPIE, Vol. 2978, San Jose, Calif., February 1997, p. 172-179).
Closed-loop biochemical microfluidic devices especially adapted to sequencing nucleic acids, as well as for high-throughput screening are described in U.S. Ser. No. 09/054,962 entitled xe2x80x9cClosed-loop Biochemical Analyzersxe2x80x9d by Knapp et al., filed Apr. 3, 1998. In brief, in the integrated systems described, it is possible to use the results of a first sequencing reaction or set of sequencing reactions to select appropriate reagents, reactants, products, or the like, for additional analysis. For example, the results of a first sequencing reaction can be used to select primers, templates or the like for additional sequencing, or to select related families of compounds for screening in high-throughput assay methods. These primers or templates are then accessed by the system and the process continues.
Although sequencing and other assay methods that utilize fluorescent markers often represent, in many ways, an improvement over methods that utilize radioactive isotopes, current fluorescent methodologies are hampered by certain deficiencies. For example, in order to identify the individual nucleotides, each nucleotide must bear a fluorescent marker that has by a unique absorbance and/or emission spectrum with a different absorbance or emission maximum. Thus, to clearly distinguish the individual nucleotides based upon the fluorescence spectrum of their tags, the absorbance or emission maxima of each tag must be clearly resolved from those of every other tag. Further, fluorescence must be monitored at a number of different wavelengths in order to detect each of the maxima and a filtering system must be employed. This is cumbersome and increases the expense of the instrumentation. This situation is additionally complicated by the dependence of the absorption or emission maxima for a compound upon the environment surrounding that compound.
Thus, a method of detecting individual fluorescently labeled compounds within a mixture of compounds which relied on a characteristic of the fluorescent moiety other than its absorption and/or emission spectrum (e.g., maxima) would represent a significant advance in the art. The present invention provides such a method.
It has now been discovered that individual members of a mixture can be distinguished and identified through the selective use of a set of fluorescent labels displaying a range of unique fluorescence lifetimes. This method is versatile and it can be practiced with a wide range of separation modalities, fluorescent markers and labeling chemistries. Further, because it detects fluorescence lifetimes, rather than fluorescence emission or excitation maxima, this method is able to resolve a mixture containing several fluorescent species with overlapping fluorescent excitation and/or emission maxima.
Thus, in a first aspect, the present invention provides a method of distinguishing between a plurality of fluorescent species. The fluorescent species are first electrokinetically transported through a microfluidic channel. The fluorescent species are then excited by irradiating them with electomagnetic energy. The excitation can occur either during the transporting or at the completion of the transporting. Following this excitation, the fluorescent molecules are allowed to return to their ground state. This process is accompanied by a fluorescence emission which is characteristic for each fluorescent species and which is characterized by a temporal duration referred to as the fluorescence lifetime.
The lifetimes for each of the fluorescent labels is detected at a detecting station and the labeled species are identified by measuring the characteristic fluorescence lifetime of the label to which they are conjugated. It will be apparent to one of skill in the art that the present method can be practiced with any of an array of detecting station configurations. The detection station can include, for example, a laser or pulse lamp to excite the fluorescent species. Additionally, any useful configuration of lenses, prisms, mirrors, diffraction gratings, monochromators and the like can be used to practice the present invention. Useful detectors include fast, high sensitivity optical detectors like PMT, Avalanche Photo Diodes and Photo Diodes. The detector can be coupled to a digital computer that receives incoming data from the detector and processes it into a form useful for distinguishing between the lifetimes of the labels.
By detecting the fluorescence emission and measuring its lifetime for each of the fluorescent species in a mixture, the different fluorescent species present in the mixture can be detected and identified. Single or overlapping emissions that are composed of species with different lifetimes can be mathematically resolved into individual lifetimes, allowing the identification of the individual fluorescent constituents contributing to the emission.
The method is generally useful for the detection and identification of a broad range of compounds. It can be used to identify individual molecules which range in size and functionality from small organic, inorganic or organometallic molecules to proteins, including enzymes, antibodies and the like. The method of the invention can also be used to characterize and identify synthetic polymers and oligomers. These polymers and oligomers find utility in diverse fields of endeavor including, industrial applications, mechanical applications, drugs, foodstuffs and textiles. Synthetic, natural and modified polymers and oligomers of biomolecules such as amino acids, nucleic acids and saccharides can also be identified using the method of the invention.
Thus, in a second aspect, the present invention provides a method of sequencing a nucleic acid polymer of interest. In this aspect of the invention, the method comprises performing a sequencing reaction on the nucleic acid polymer. Any of the sequencing reactions known in the art is appropriate for use in this aspect. Thus, methods which chemically or enzymatically degrade or synthesize nucleic acids are of use in practicing the present invention.
During the course of the sequencing reaction, one or more fluorescent labels is incorporated into either the nucleic acid being sequenced or a sequence complementary to the nucleic acid being sequenced. Several methods for performing this incorporating are known in the art. A non-limiting list includes the Sanger, Sanger dideoxy and Maxam-Gilbert sequencing methodologies.
Sequencing reaction mixtures that are useful in practicing the present invention include those that contain the nucleic acid to be sequenced and a fluorescent label. The fluorescent label is attached to a first labeled nucleic acid selected from the group consisting of labeled nucleic acids, labeled nucleic acid polymers and combinations thereof. The fluorescent species are electrokinetically transported through a microfluidic channel to resolve or partially resolve the mixture into separate components.
As discussed above, the fluorescent label will, following excitation, emit electromagnetic energy that is characterized by a distinct and detectable lifetime. When more than one fluorescent label is utilized in the sequencing reaction mixture, each of the labels will have a fluorescent lifetime that is distinct from other labels and thereby detectable. The fluorescence emission is detected at a detecting station.
In addition to the above-described methods, the present invention also provides an apparatus that is particularly useful in practicing the methods of this invention. The apparatus is capable of distinguishing between a plurality of fluorescent species, wherein each of the fluorescent species has a fluorescence emission, the emission having a characteristic fluorescence lifetime.
The apparatus of the invention comprises a microfluidic device that contains at least one microchannel therein. The fluorescent species flows through the microchannel by means of, for example, electroosmosis, electrokinesis, capillarity and the like. The microchannel is linked to a detecting station that is capable of detecting the fluorescent species in the microchannel. The signal from the detector is sent to a digital computer that is operably linked to the detector. The digital computer is appropriately configured or programmed to determine the fluorescence lifetimes of the fluorescent species.
Other objects and advantages of the present invention will be apparent from the detailed description that follows.